{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T20:44:50Z","timestamp":1778618690428,"version":"3.51.4"},"reference-count":29,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2019,1,24]],"date-time":"2019-01-24T00:00:00Z","timestamp":1548288000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2019,1,24]],"date-time":"2019-01-24T00:00:00Z","timestamp":1548288000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The BRAF inhibitors dabrafenib and vemurafenib induce remarkable clinical responses in patients with BRAF-mutated melanomas. However, adverse events, including the emergence of secondary tumors and drug resistance, have been reported. Studies have revealed that undesirable RAF dimerization induced by inhibitors promotes these adverse effects. Here, we developed highly sensitive biosensors of RAF dimerization in cells utilizing the split enhanced click beetle luciferase (Emerald Luc,\u00a0ELuc) complementation technique. We demonstrated that our biosensor system works effectively for high-throughput screens in the microplate format. A comprehensive analysis of commercially available RAF inhibitors performed using this assay system revealed that the inhibitors exhibit various potencies in inducing the dimerization of RAF isoforms, and their dimerization potencies do not always correlate with the RAF enzyme inhibition. This sensitive assay system will become a powerful tool to discover next-generation BRAF inhibitors with safer profiles.<\/jats:p>","DOI":"10.1038\/s41598-018-37213-2","type":"journal-article","created":{"date-parts":[[2019,1,24]],"date-time":"2019-01-24T11:04:17Z","timestamp":1548327857000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Development of Highly Sensitive Biosensors of RAF Dimerization in Cells"],"prefix":"10.1038","volume":"9","author":[{"given":"Kyoko","family":"Miyamoto","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0077-2188","authenticated-orcid":false,"given":"Masaaki","family":"Sawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2019,1,24]]},"reference":[{"key":"37213_CR1","doi-asserted-by":"publisher","first-page":"928","DOI":"10.1038\/nrd4281","volume":"13","author":"AA Samatar","year":"2014","unstructured":"Samatar, A. A. & Poulikakos, P. I. Targeting RAS\u2013ERK signalling in cancer: promises and challenges. Nat. Rev. Drug Discov. 13, 928\u2013942 (2014).","journal-title":"Nat. Rev. Drug Discov."},{"key":"37213_CR2","doi-asserted-by":"crossref","unstructured":"Karoulia, Z., Gavathiotis, E. & Poulikakos, P. I. New perspectives for targeting RAF kinase in human cancer. Nat. Rev. Cancer 17,\u00a0676\u2013691\u00a0(2017).","DOI":"10.1038\/nrc.2017.79"},{"key":"37213_CR3","doi-asserted-by":"publisher","first-page":"180","DOI":"10.4161\/sgtp.26117","volume":"4","author":"AK Freeman","year":"2013","unstructured":"Freeman, A. K., Ritt, D. A. & Morrison, D. K. The importance of Raf dimerization in cell signaling. Small GTPases 4, 180\u20135 (2013).","journal-title":"Small GTPases"},{"key":"37213_CR4","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1038\/nrm3979","volume":"16","author":"H Lavoie","year":"2015","unstructured":"Lavoie, H. & Therrien, M. Regulation of RAF protein kinases in ERK signalling. Nat. Rev. Mol. Cell Biol. 16, 281\u2013298 (2015).","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"37213_CR5","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1016\/j.cell.2009.12.040","volume":"140","author":"SJ Heidorn","year":"2010","unstructured":"Heidorn, S. J. et al. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. Cell 140, 209\u2013221 (2010).","journal-title":"Cell"},{"key":"37213_CR6","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1038\/nature08902","volume":"464","author":"PI Poulikakos","year":"2010","unstructured":"Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427\u2013430 (2010).","journal-title":"Nature"},{"key":"37213_CR7","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1038\/nature08833","volume":"464","author":"G Hatzivassiliou","year":"2010","unstructured":"Hatzivassiliou, G. et al. LETTERS RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431\u2013435 (2010).","journal-title":"Nature"},{"key":"37213_CR8","doi-asserted-by":"publisher","first-page":"485","DOI":"10.1016\/j.ccell.2016.06.024","volume":"30","author":"Z Karoulia","year":"2016","unstructured":"Karoulia, Z. et al. An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling. Cancer Cell 30, 485\u2013498 (2016).","journal-title":"Cancer Cell"},{"key":"37213_CR9","doi-asserted-by":"publisher","first-page":"2516","DOI":"10.1021\/ac0013296","volume":"73","author":"T Ozawa","year":"2001","unstructured":"Ozawa, T., Kaihara, A., Sato, M., Tachihara, K. & Umezawa, Y. Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal. Chem. 73, 2516\u20132521 (2001).","journal-title":"Anal. Chem."},{"key":"37213_CR10","doi-asserted-by":"publisher","first-page":"4176","DOI":"10.1021\/ac0300800","volume":"75","author":"A Kaihara","year":"2003","unstructured":"Kaihara, A., Kawai, Y., Sato, M., Ozawa, T. & Umezawa, Y. Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal. Chem. 75, 4176\u20134181 (2003).","journal-title":"Anal. Chem."},{"key":"37213_CR11","doi-asserted-by":"publisher","first-page":"2552","DOI":"10.1021\/ac100104q","volume":"82","author":"N Misawa","year":"2010","unstructured":"Misawa, N. et al. Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: An approach to the study of G-protein-coupled receptors. Anal. Chem. 82, 2552\u20132560 (2010).","journal-title":"Anal. Chem."},{"key":"37213_CR12","doi-asserted-by":"publisher","first-page":"384","DOI":"10.1016\/j.ccell.2015.08.002","volume":"28","author":"S Peng","year":"2015","unstructured":"Peng, S. et al. Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers Article Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers. Cancer Cell 28, 384\u2013398 (2015).","journal-title":"Cancer Cell"},{"key":"37213_CR13","doi-asserted-by":"crossref","unstructured":"Nakajima, Y. et al. Enhanced beetle luciferase for high-resolution bioluminescence imaging. PLoS One 5, e10011 (2010).","DOI":"10.1371\/journal.pone.0010011"},{"key":"37213_CR14","doi-asserted-by":"publisher","first-page":"542","DOI":"10.1038\/nature08314","volume":"461","author":"T Rajakulendran","year":"2009","unstructured":"Rajakulendran, T., Sahmi, M., Lefran\u00e7ois, M., Sicheri, F. & Therrien, M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, 542\u2013545 (2009).","journal-title":"Nature"},{"key":"37213_CR15","doi-asserted-by":"publisher","first-page":"428","DOI":"10.1038\/nchembio.1257","volume":"9","author":"H Lavoie","year":"2013","unstructured":"Lavoie, H. et al. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat. Chem. Biol. 9, 428\u2013436 (2013).","journal-title":"Nat. Chem. Biol."},{"key":"37213_CR16","doi-asserted-by":"publisher","first-page":"7043","DOI":"10.1158\/0008-5472.CAN-13-1825","volume":"73","author":"A Nakamura","year":"2013","unstructured":"Nakamura, A. et al. Antitumor activity of the selective pan-RAF inhibitor TAK-632 in BRAF inhibitor-resistant melanoma. Cancer Res. 73, 7043\u20137055 (2013).","journal-title":"Cancer Res."},{"key":"37213_CR17","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/nature14982","volume":"526","author":"C Zhang","year":"2015","unstructured":"Zhang, C. et al. RAF inhibitors that evade paradoxical MAPK pathway activation. Nature 526, 583\u2013586 (2015).","journal-title":"Nature"},{"key":"37213_CR18","doi-asserted-by":"crossref","unstructured":"Lyons, J. F., Wilhelm, S., Hibner, B. & Bollag, G. Discovery of a novel Raf kinase inhibitor. Endocrine-Related Cancer 8, 219\u2013225 (2001).","DOI":"10.1677\/erc.0.0080219"},{"key":"37213_CR19","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1002\/ijc.25864","volume":"129","author":"SM Wilhelm","year":"2011","unstructured":"Wilhelm, S. M. et al. Regorafenib (BAY 73-4506): A new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int. J. Cancer 129, 245\u2013255 (2011).","journal-title":"Int. J. Cancer"},{"key":"37213_CR20","doi-asserted-by":"publisher","first-page":"11100","DOI":"10.1158\/0008-5472.CAN-06-2554","volume":"66","author":"AJ King","year":"2006","unstructured":"King, A. J. et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 66, 11100\u201311105 (2006).","journal-title":"Cancer Res."},{"key":"37213_CR21","doi-asserted-by":"crossref","unstructured":"Elenbaas, B. et al. BIIB024, a potent pan-Raf kinase inhibitor for melanoma and solid tumors. Eur. J. Cancer Suppl. 8, 40\u201341\u00a0(2010).","DOI":"10.1016\/S1359-6349(10)71810-3"},{"key":"37213_CR22","doi-asserted-by":"publisher","first-page":"3041","DOI":"10.1073\/pnas.0711741105","volume":"105","author":"J Tsai","year":"2008","unstructured":"Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. 105, 3041\u20133046 (2008).","journal-title":"Proc. Natl. Acad. Sci."},{"key":"37213_CR23","doi-asserted-by":"publisher","first-page":"e67583","DOI":"10.1371\/journal.pone.0067583","volume":"8","author":"AJ King","year":"2013","unstructured":"King, A. J. et al. Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF\/MEK Tool Combination Reduced Skin Lesions. PLoS One 8, e67583 (2013).","journal-title":"PLoS One"},{"key":"37213_CR24","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.cca.2017.07.012","volume":"472","author":"M Rousset","year":"2017","unstructured":"Rousset, M. et al. Trough dabrafenib plasma concentrations can predict occurrence of adverse events requiring dose reduction in metastatic melanoma. Clin. Chim. Acta 472, 26\u201329 (2017).","journal-title":"Clin. Chim. Acta"},{"key":"37213_CR25","doi-asserted-by":"crossref","first-page":"1470","DOI":"10.1093\/annonc\/mdv189","volume":"26","author":"E Funck-Brentano","year":"2015","unstructured":"Funck-Brentano, E. et al. Plasma vemurafenib concentrations in advanced BRAFV600mut melanoma patients: impact on tumour response and tolerance. Ann. Oncol. 26, 1470\u20131475 (2015).","journal-title":"Ann. Oncol."},{"key":"37213_CR26","doi-asserted-by":"publisher","first-page":"43458","DOI":"10.18632\/oncotarget.16465","volume":"8","author":"H Mai","year":"2017","unstructured":"Mai, H. et al. In-vivo relation between plasma concentration of sorafenib and its safety in Chinese patients with metastatic renal cell carcinoma: a single-center clinical study. Oncotarget 8, 43458\u201343469 (2017).","journal-title":"Oncotarget"},{"key":"37213_CR27","doi-asserted-by":"crossref","unstructured":"Yang, N. J. & Hinner, M. J. Getting Afross the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins.\u00a0Site-Specific Protein Labeling. Methods Mol. Biol. 1266,\u00a029\u201353 (2015).","DOI":"10.1007\/978-1-4939-2272-7_3"},{"key":"37213_CR28","doi-asserted-by":"crossref","unstructured":"Zimmermann, S. et al. MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src. Oncogene 15, 1503\u20131511 (1997).","DOI":"10.1038\/sj.onc.1201322"},{"key":"37213_CR29","doi-asserted-by":"publisher","first-page":"425","DOI":"10.1242\/jcs.036319","volume":"122","author":"S-Y Shin","year":"2009","unstructured":"Shin, S.-Y. et al. Positive- and negative-feedback regulations coordinate the dynamic behavior of the Ras-Raf-MEK-ERK signal transduction pathway. J. Cell Sci. 122, 425\u201335 (2009).","journal-title":"J. Cell Sci."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-37213-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-37213-2","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-37213-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,17]],"date-time":"2022-12-17T05:17:42Z","timestamp":1671254262000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-37213-2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,1,24]]},"references-count":29,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["37213"],"URL":"https:\/\/doi.org\/10.1038\/s41598-018-37213-2","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,1,24]]},"assertion":[{"value":"27 July 2018","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"30 November 2018","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 January 2019","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"K.M. is an employee of CarnaBio USA, Inc. M.S. is Chief Scientific Officer of CarnaBio USA, Inc. and Carna Biosciences, Inc. The authors declare no conflict of non-financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"636"}}